Figure 3.

Methods of measuring extracellular diffusion coefficients and other important ECS parameters in brain tissue. Real-time iontophoresis is a technique that employs iontophoresis of small ions, typically the 74 Da tetramethylammonium (TMA) cation, from a source microelectrode and the subsequent measurement of the resulting local concentration over time some short distance away from the release site by an ion-selective microelectrode. The real-time iontophoretic technique is useful because it potentially can be applied to any brain region, although it has typically been limited to small, charged ions for which suitable ion-selective microelectrodes can be fabricated. Particular strengths of the real-time iontophoretic method are that it can be performed repeatedly in the same animal at different locations/time points and it yields the ECS volume fraction (α) and clearance/uptake information in addition to diffusion coefficients. The ventriculo-cisternal perfusion technique is used to measure transport into brain areas bordering the ventricular system (a modification of the method may also be used to measure transport across the brain’s pial surface from the subarachnoid space). Typically, a radiotracer is infused into the cerebrospinal fluid (CSF) of the lateral ventricle and then removed from the cistern at an equivalent rate. After a suitable time, the brain is carefully removed and samples are taken to measure the radiotracer concentration at various depths; the resulting distribution may then be analyzed to yield 3, clearance information and diffusion coefficients. While the range of substances that may be explored using the ventriculo-cisternal perfusion method is broad, the complexity of the surgical preparation and difficulties associated with utilizing a radioactivity-based technique has limited its contemporary use; it also is only capable of producing a single time point per animal. Finally, integrative optical imaging is a technique in which a very small volume of fluid containing a fluorescently labeled substance is pressure-injected into the brain and the resulting concentration distribution is recorded over time using an epifluorescent microscope and CCD camera. Diffusion coefficients are obtained by fitting fluorescence intensity curves from the resulting images with the appropriate solution to the diffusion equation. While integrative optical imaging cannot easily be used to obtain 3 or clearance/uptake information, it is routinely employed to measure diffusion coefficients for a wide range of fluorescent substances and fluorophore-drug conjugates at multiple locations and time points within the same animal.